Immunogenic agents against Burkholderia pseudomallei and/or Burkholderia mallei, comprising lipopolysaccharide, capsular polysaccharide and/or proteins from Burkholderia pseudomallei

ABSTRACT

An immunogenic agent which comprises a killed strain of  Burkholderia pseudomallei , or a combination of components thereof which combination produces a protective immune response in an animal to whom it is administered, and which comprises at least two members selected from the group consisting of (i) a lipopolysaccharide of  Burkholderia pseudomallei , (ii) a capsular polysaccharide of  Burkholderia pseudomallei  and (iii) a protein of  Burkholderia pseudomallei  or an immunogenic variant thereof or an immunogenic fragment of either of these, or a nucleic acid which expresses said protein, immunogenic variant or immunogenic fragment thereof in a host animal; for use in the prevention or treatment of infection by  Burkholderia pseudomallei  and/or  Burkholderia mallei.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/088,748, filed Oct. 28, 2008, and issued as U.S. Pat. No. 7,955,601on Jun. 7, 2011, which is the U.S. national phase of InternationalApplication No. PCT/GB2006/003628, filed Oct. 2, 2006, and published inEnglish on Apr. 5, 2007, as International Publication No. WO2007/036735, which application claims priority to Great BritainApplication No. 0519871.8, filed Sep. 30, 2005. The contents of each ofthe foregoing application are incorporated herein by reference in theirentirety.

The present invention relates to immunogenic agents which are useful asprophylactic or therapeutic vaccines against infection by Burkholderiapseudomallei and/or Burkholderia mallei.

Burkholderia pseudomallei is the causative agent of melioidosis, asevere disease of humans and animals. The bacterium is present in theenvironment, mainly in South East Asia, Northern Australia, parts ofAfrica, South and Central America. Although melioidosis has historicallybeen considered to be a relatively rare disease it is being diagnosed inan increasing number of countries and with an increasing frequency. Thisis probably due to a combination of factors, such as recent improvementsin diagnostic techniques, a greater awareness of the disease and anincrease in global travel from areas of the world where melioidosis isendemic.

Melioidosis can present in a number of forms which have been describedas acute septicaemic, acute pulmonary, sub-acute and chronic diseases.In some cases a persistent sub-clinical infection is established withthe subsequent ability to become septicaemic. The factors whichinfluence the outcome of disease are not known, although it has beensuggested that differences in the virulence of different strains mightcontribute to the clinical outcome of disease. In addition, melioidosisis most frequently seen in diabetics, those with impaired cellularimmunity or those with a history of drug or alcohol abuse, suggestingthat differences in the immunological status of the host might alsoinfluence the outcome of the disease.

Currently no vaccine exists to protect against melioidosis. B.pseudomallei has previously been shown to produce two types oflipopolysaccharide (LPS), termed OPSI and OPSII, and a capsularpolysaccharide [Y. Isshiki et al, FEMS Microbiol. Lett. 2001, 199,21-25, S. Reckseidler et al. Infect. Immun. 2001, 69(1) 34-44].

The LPS of B. pseudomallei has been shown to be biologically active andcapable of stimulating murine macrophages [M. Matsuura et al. FEMSMicrobiol. Lett 1996, 137, 79-83]. Further studies have shown that LPSis capable of stimulating an immune response in the murine model ofdisease. Polyclonal antisera raised against the LPS was found to bepassively protective against challenge with B. pseudomallei [M. Nelsonet al. Journal of Medical Microbiology 2004, 53 (12) 1177-1182].Conjugates of LPS and B. pseudomallei flagellin have been proposed andevaluated as putative vaccine candidates [P. Brett et al. Infect Immun.1996, 64(7) 2824-2828].

There is also evidence that capsular polysaccharides play a role in thevirulence of both Gram-negative and Gram-positive bacteria and areimportant in protecting the bacteria from host defense systems.Insertional inactivation of the capsule biosynthetic pathway results inthe production of an avirulent B. pseudomallei strain [Reckseidler et alsupra., Atkins et al. Journal Medical Microbiology 2002, 51, 539-547].Capsular polysaccharide is produced by both B. pseudomallei strainsK96243 and 576, and has been shown to be a potential vaccine candidate[Nelson et al. supra].

It has been previously demonstrated that passive administration ofmonoclonal antibody raised against protein antigens of B. pseudomalleican confer protection [S. M. Jones et al. Journal of MedicalMicrobiology 2002, 51 (12) 1055-1062].

Although antisera raised against these conjugate vaccines is protectivein animal studies, active immunisation with the conjugate has not beenreported.

Inactivated vaccines have been used previously to provide protectionagainst a number of diseases including typhoid, whooping cough, polioand rabies and demonstrate that vaccination against individual diseasesis possible given the correct stimulation of the immune system byantigen. However, it is not possible to predict whether, in anyparticular case, such preparations will provide the sort of stimulation,which would lead to protection, and there have been many instances wherekilled whole cell vaccines have proved ineffective.

For instance, killed whole-cell preparations of mycobacterium havehistorically been regarded as inefficient vaccines against for exampleTB, and so the live attenuated vaccine, bacilli Calmette-Guerin (BCG) isthe registered vaccine. Killed whole cell vaccine against Yersiniapestis has been found to offer poor protection against pneumonic disease(R. W. Titball et al. Vaccine 2001, 19(30) 4175-84. A recent study usinga killed whole-cell Lishmania amazonensis vaccine has shown that itprovides no protection against disease (I. D. Velez et al., Trans. R SocTrop. Med Hyg, 2005, 99(8):593-8).

Generally, vaccines which induce specifically antibody responses such askilled whole cell vaccines are regarded as being ineffective againstintracellular pathogens such as Burkholderia.

However, the applicants investigated whether immunisation with killedwhole cells of B. pseudomallei can protect mice against experimentalmelioidosis, and found good results.

The model used provided for an assessment of the relative roles ofcapsule, LPS and surface proteins in protection against disease, and soprovide improved conjugate vaccines also.

According to the present invention, there is provided an immunogenicagent which comprises a killed strain of Burkholderia pseudomallei, or acombination of components thereof which combination produces aprotective immune response in an animal to whom it is administered, andwhich comprises at least two members selected from the group consistingof (i) a lipopolysaccharide of Burkholderia pseudomallei, (ii) acapsular polysaccharide of Burkholderia pseudomallei and (iii) animmunogenic protein of Burkholderia pseudomallei or an immunogenicvariant thereof or an immunogenic fragment of either of these, or anucleic acid which expresses said protein, immunogenic variant orimmunogenic fragment in a host animal; for use in the prevention ortreatment of infection by Burkholderia pseudomallei and/or Burkholderiamallei.

Immunogenic agents according to the invention have been found to providegood protection against challenge by B. pseudomallei species in animalmodels, and can therefore form the basis of prophylactic or therapeuticvaccines in animals such as humans.

Preferably, the protective response is protective for at least 20 dayspost-challenge or infection by Burkholderia pseudomallei and/orBurkholderia mallei.

In a particular embodiment of the invention, the immunogenic agent is akilled strain of Burkholderia pseudomallei, preferably one whichincludes a capsular polysaccharide.

In one aspect of the invention, the killed strain of Burkholderiapseudomallei produces a protective immune response in an animal to whomit is administered. Preferably, the protective response is protectivefor at least 20 days post-challenge or infection by Burkholderiapseudomallei and/or Burkholderia mallei.

The strain may be killed by conventional methods for example by heattreatment, freeze-thaw treatment, sonication, sudden pressure drop ortreatment using an inactivating agent such as formalin, azide, sodiumhypochlorite, phenol, saponin, detergent (such as non-ionic detergent)lysozyme, propiolactone and in particular betapropiolactone, binaryethyleneimine (U.S. Pat. No. 5,565,205) or Thimerosal (U.S. Pat. No.5,338,543). Preferably the strain is killed using a treatment whichleaves at least some surface molecules intact.

In particular, however, the immunogenic agent is a heat-killed strain.

It is suitably prepared by heating cultures of B. pseudomallei totemperatures of from 50-90° C. for a period sufficient to ensure thatall cells are inactive. This can be tested using routine methods asillustrated hereinafter. In particular, heating a culture of B.pseudomallei to a temperature of about 80° C. for a period of about 3hours has been found to result in complete inactivation.

Suitable strains include any of the available strains irrespective ofthe lipopolysaccharide serotype. Examples of known strains include B.pseudomallei K96243 (Proc. Natl. Acad. Sci. USA (2004) 01 (39)14240-14245) and B. pseudomallei 576. Examples are B. pseudomalleistrains are available for example from the National Collection of TypeCultures, Central Public Health Laboratory, 61 Colindale Avenue, LondonNW9, 5HT UK where examples include those stored as NCTC 4845, NCTC12939, NCTC13177, NCTC13178 and NCTC 13172, but others may be isolatedfrom natural sources for example from patients suffering from B.pseudomallei infection, or from environmental sources such as soilsamples.

In one embodiment the strain is one which has an atypical LPS serotype(OPSII), such as B. pseudomallei 576.

In another embodiment, the strain is one which has a typical LPSserotype (OPSI) such as B. pseudomallei K96243, the genome sequence ofwhich is available.

In an alternative embodiment of the invention, the immunogenic agent isa combination of Burkholderia pseudomallei components comprising atleast two members of the group selected from the group consisting of (i)a lipopolysaccharide of Burkholderia pseudomallei, (ii) a capsularpolysaccharide of Burkholderia pseudomallei and (iii) an immunogenicprotein of Burkholderia pseudomallei or an immunogenic variant thereof,or an immunogenic fragment of either of these, or a nucleic acid whichexpresses said immunogenic protein, variant or fragment in a hostanimal.

It has been found that this group of components can act synergisticallytogether, enhancing the protective effect that would be obtained usingthe components individually.

In one embodiment, the combination comprises a lipopolysaccharide and acapsular polysaccharide of Burkholderia pseudomallei.

In a further embodiment, the combination comprises a lipopolysaccharideand an immunogenic protein of Burkholderia pseudomallei or animmunogenic variant thereof, or an immunogenic fragment of either ofthese, or a nucleic acid which expresses said immunogenic protein,variant or fragment in a host animal.

Preferably all three components are present and so the immunogenic agentcomprises (i) a lipopolysaccharide of Burkholderia pseudomallei, (ii) acapsular polysaccharide of Burkholderia pseudomallei and (iii) animmunogenic protein of Burkholderia pseudomallei or an immunogenicvariant thereof, or an immunogenic fragment of either of these, or anucleic acid which expresses said immunogenic protein, variant orfragment in a host animal.

As used herein, the expression “variant” refers to sequences of aminoacids which differ from the base sequence from which they are derived inthat one or more amino acids within the sequence are substituted forother amino acids, but which retain the ability of the base sequence toproduce an immunogenic response which recognises epitopes of B.pseudomallei. Amino acid substitutions may be regarded as “conservative”where an amino acid is replaced with a different amino acid with broadlysimilar properties. Non-conservative substitutions are where amino acidsare replaced with amino acids of a different type. Broadly speaking,fewer non-conservative substitutions will be possible without alteringthe biological activity of the polypeptide. Suitably variants will be atleast 70% identical, for instance at least 75% identical, especially atleast 80% identical. In particular variants will be at least 90%identical, and preferably at least 95% identical to the base sequence.

Identity in this instance can be judged for example using the BLASTprogram or the algorithm of Lipman-Pearson, with Ktuple:2, gappenalty:4, Gap Length Penalty:12, standard PAM scoring matrix (Lipman,D. J. and Pearson, W. R., Rapid and Sensitive Protein SimilaritySearches, Science, 1985, vol. 227, 1435-1441).

The term “fragment” refers to any portion of the given amino acidsequence, which includes an epitope and so has immunogenic activity.Fragments will suitably comprise at least 5 and preferably at least 10consecutive amino acids from the basic sequence.

The combination may comprise more than one lipopolysaccharide, and in aparticularly embodiment, it will contain a B. pseudomalleilipopolysaccharide of each of the two known serotypes, OPSI and OPSII asdescribed above.

Where the combination includes component (iii) above, this is suitablyone or more immunogenic proteins of Burkholderia pseudomallei or animmunogenic variant thereof, or an immunogenic fragment of either ofthese. Preferably component (iii) comprises one or more immunogenicproteins of Burkholderia pseudomallei.

Suitable proteins are in particular surface proteins. Thus in aparticular embodiment, the combination will comprise an immunogenicsurface protein of Burkholderia pseudomallei or an immunogenic variantthereof, or an immunogenic fragment of either of these, or a nucleicacid which expresses said immunogenic surface protein, variant orfragment in a host animal

Particular surface proteins include ABC transporter proteins (forexample as described in copending British Patent Application No.0507632.8) porins, pili, adhesins, ion acquisition proteins, orcomponents of the type 3 secretion system.

The immunogenicity of any particular protein can be determined usingroutine methods as would be apparent to the skilled person.

The selection of specific proteins for testing in this way mayalternatively be determined by examination of the proteome of the B.pseudomallei species, derivable from the known genomic sequences, usingfor instance the method described in WO 03/073351.

Alternatively, B. pseudomallei proteins can be made or isolated andtested using routine methods to ensure that they are immunogenic.

In a particularly preferred embodiment, the combination comprises morethan one such protein or immunogenic variant, or fragment of either ofthese.

In an alternative embodiment, the combination comprises a nucleic acidwhich expresses said immunogenic protein, variant or fragment in a hostanimal. In this instance, the nucleic acid might be incorporated into anexpression vector, in particular a pharmaceutically acceptableexpression vector such as a viral vector such as vaccinia (for instance,the Lister strain), or adenovirus vectors which are suitably attenuated,or a bacterial expression vector such as an attenuated Salmonella strainfor instance attenuated strains of S. typhi or S. typhimurium such asSL3261. Nucleic acids may also be in the form of a plasmid or “nakedDNA” vaccine. Suitable plasmids include those known in the art and manyare commercially available.

Immunogenic agents of the invention are suitably administered in theform of one or more pharmaceutical compositions, which suitably furthercomprises a pharmaceutically acceptable carrier.

Where the immunogenic agent comprises a combination as described above,individual components may be administered separately or together, butare suitable formulated together in a single dosage unit.

The nature of the carrier will vary depending upon the nature of theimmunogenic agent, the mode of administration selected etc. inaccordance with normal pharmaceutical procedure.

Suitable carriers are well known in the art and include solid and liquiddiluents, for example, water, saline or aqueous ethanol. The liquidcarrier is suitably sterile and pyrogen free.

The compositions may be suitable for oral use (for example as tablets,lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions,dispersible powders or granules, syrups or elixirs), for administrationby inhalation (for example as a finely divided powder or a liquidaerosol), for administration by insufflation (for example as a finelydivided powder) or for parenteral administration (for example as asterile aqueous or oily solution for intravenous, subcutaneous,intramuscular or intramuscular or intradermal dosing) or as asuppository for rectal dosing.

They will be combined with pharmaceutically acceptable excipients, suchas inert diluents, granulating or disintegrating agents, binding agents,lubricating agents, preservative agents and anti-oxidants. Tabletformulations may be uncoated or coated either to modify theirdisintegration and the subsequent absorption of the active ingredientwithin the gastrointestinal track, or to improve their stability and/orappearance, in either case, using conventional coating agents andprocedures well known in the art.

Where the immunogenic agent includes a nucleic acid element which is inthe form of “live vaccine”, this will be formulated to ensure that theyproduce the desired effect. For example, where the vaccine comprises aviral vector, they may be contained within formulations suitable forparenteral administration or, when possible, for oral administration,inhalation or insufflation.

Where bacterial vectors such as attenuated Salmonella strains are usedto deliver the nucleic acid which encodes a protein element of theimmunogenic agent, they are suitably formulated for oral administration.

In particular, where the vaccine comprises a naked DNA vaccine, theywill be formulated such that they are suitable for parenteraladministration, for example by combination with liquids such as saline.These compositions are preferably formulated for intramuscularinjection, although other means of application are possible as describedin the pharmaceutical literature, for example administration using aGene Gun, (Bennett et al., (2000), Vaccine 18, 1893-1901). Oral orintra-nasally delivered formulations are also possible. Suchformulations include delivery of the plasmid DNA via a bacterial vectorsuch as species of Salmonella or Listeria (Sizemore et al (1997).Vaccine 15, 804-807).

Dosages of the vaccine used in any particular case will depend uponfactors such as the particular protein used or expressed by the vaccine,the nature of the patient receiving the treatment etc. and will bedetermined in any particular case in accordance with conventionalclinical practice. Generally speaking however, in general, theimmunogenic agent will be administered in an amount of from 0.5 mg to 75mg per kg body weight. Where the immunogenic includes a “live” vaccinecomponent, such as a virus vector, dosages of the vector may be in therange of from 10⁴-10¹² pfu (pfu=particle forming units).

The compositions of the invention may further additional other activecomponents. For example, the other component may comprise an adjuvantwhich enhances the host's immune response, and/or the polypeptide may becombined with an antigen giving protective immunity against a differentpathogen to form a multivalent vaccine in order to increase thebenefit-to-risk ratio of vaccination.

In a particularly preferred embodiment, the other active componentcomprises an adjuvant which enhances the host's immune response and inparticular promotes a cellular immune response, such as a CD8+, a CD4+and/or a Th1 response.

Adjuvants which may achieve these effects include cytokines such asinterleukins and interferons. In particular, the other componentcomprises a cytokine such as an interleukin, which acts as a Th-1adjuvant. A particularly preferred interleukin for inclusion in thevaccines of the invention is IL-12, which has been shown to drive theexpansion of a protective Th-1 cell response during early murinetularemia (Golovliov I, et al. (1995). Infection and Immunity63(2):534-8).

Other types of pharmaceutically adjuvant include Freund's incompleteadjuvant, aluminium compounds such as aluminium hydroxide, polycationiccarbohydrates such as chitosan and derivatives thereof, for example asdescribed in WO00/56362, or adjuvants described in WO00/56361 orWO00/56282.

However, for reasons discussed in more detail below, a particularlypreferred class of adjuvants are those which activate or stimulateantigen-presenting cells, such as dendritic cells. These can evoke inparticular long lasting protective immune responses against a range ofstrains. Examples of such adjuvants are Toll-like receptor (TLR) ligandssuch as CpG oligonucleotides (for example as described in U.S. Pat. No.6,429,199), bacterial lipopolysaccharides or lipoproteins. Where theimmunogenic reagent includes as a component a lipopolysaccharide derivedfrom B. pseudomallei, the Toll-like receptor ligand used as theadjuvant, is suitably other than a lipopolysaccharide derived from B.pseudomallei.

These are suitably coadministered or even linked to the immunogenicreagents described above, which may in particular be recombinant B.pseudomallei proteins, in order to enhance the response of the hostsantigen-presenting cells, so as to produce synergistic levels ofprotection.

In a further particular embodiment however, the immunogenic agentdescribed above may be combined with antigen-presenting cells. Thiscombination is then co-administered.

Antigen-presenting cells are instrumental in producing an immuneresponse and operate by various mechanisms. They include macrophages anddendritic cells.

Dendritic cells (DC's) are specialised antigen presenting cells thathave a central role in initiating T-cell responses. Immature DC's engulfpathogens, initiating a process of maturation, which includes theirmigration to lymphoid organs and culminates in enhanced expression ofMHC II—peptide complexes and various co-stimulatory molecules. Theyconvey information regarding the nature of the microbial stimulus toT-cells and direct the development of polarised T-cell responses alongeither the type 1 or type 2 pathways.

The applicants have found that cultured dendritic cells pulsed withimmunogenic reagents described above, and in particular killed B.pseudomallei strains such as heat killed B. pseudomallei 4845 or K96243can be used to immunise animals and evoke both cell mediated and humoralimmune responses in the recipients. This gives rise to ex-vivotherapeutic options.

As used herein the term “ex-vivo” describes a procedure that is carriedout on a sample taken from a patient, the sample being returned to thepatient after treatment.

In a particular embodiment therefore, the invention provides animmunogenic reagent as described above for use in the preparation of amedicament for administration to antigen-presenting cells (APC) of apatient, for the activation of the immune response of said patient.

Compositions comprising these immunogenic reagents andantigen-presenting cells such as dendritic cells are also novel and forma further aspect of the invention.

Adjuvants such as CpG oligodeoxynucleotides (unmethylatedcytidine-guanosine dinucleotides flanked by patterns of bases asdescribed for example in U.S. Pat. No. 6,429,199) can be used either toenhance APC and particularly DC maturation in vitro or as an adjuvantwhen administered to animals at the time they are immunised with antigenpulsed DC. In vitro and in vivo exposure of DC to CpG causesupregulation of MHC II and expression of the co-stimulatory moleculesCD40, CD80 and CD86.

Cohorts of animals immunised with dendritic cells were taken forward forvirulent challenge with different strains of B. pseudomallei andprotection against parenteral challenge was demonstrated.

An ex-vivo medicament of this type will advantageously allow theactivation of antigen presenting cells to be controlled and monitored,thus providing a patient with transfected cells which will increase thepatients immune response whilst also removing existing problems withvaccines such as dilution in body fluids and chemical and enzymaticdegradation in vivo.

The ability of DC pulsed with heat killed B. pseudomallei to induceproliferation of naïve mouse spleen cells has been demonstrated asillustrated below. The magnitude of proliferative responses was muchhigher in animals which had been immunised with antigen pulsed DC. Thisdata shows that DC immunisation has the potential to induce antigenspecific memory immune responses in recipient animals. Additionally,significantly increased numbers of interferon-γ producing cells inanimals immunised with CpG treated heat killed B. pseudomallei pulsed DCcorrelates with data from other studies involving the use of CpG ODN inwhich enhanced IFN-γ production has been seen.

Elevated levels of IFN-γ correlate with protection against challenge inmurine models of melioidosis and the use of CpG-matured, B. pseudomalleipulsed DC was found to enhance IFN-γ production and also to result insignificantly enhanced protection.

Low titres of antigen specific IgG in serum of vaccines were found butthis may have been due to the very small amounts of antigen delivered byDC immunisation, as it is known that relatively large amounts of antigenare required in order for a robust antibody response to be mounted. Theuse of CpG either in DC maturation or as an adjuvant at the point of DCimmunisation increased antibody titres significantly compared with thenon-CpG treated group, as has been seen in other studies. This couldhave contributed to the robust protection seen in the CpG treatedgroups, as antibody may have an important role in defense againstmelioidosis, with high circulating titres of antigen specific monoclonalantibody being shown to provide protection against challenge.

The importance of antibody is likely to be limited to the early stagesof an infection however, before the bacteria are able to gain access tothe intra-cellular niche in which they are known to thrive. Once thebacteria have established an intra-cellular infection cell mediatedimmune mechanisms are required for efficient eradication of thepathogen.

A DC immunisation strategy is able to evoke cell-mediated immunemechanisms, as evidenced by increased IFN-γ production and proliferationof spleen cells in response to antigen stimulation in vitro, and theseeffectors were clearly contributing to the high levels of challengesurvival, which the applicants found.

When animals were immunised with DC matured in the presence of CpG 1826and then challenged with B. pseudomallei K96243 there were 9 of 10survivors, while when CpG 1826 was used as an adjuvant there were 7 of10 survivors. These survival rates are significantly better than thoseseen when control non-CpG ODN was used to mature the DC's (3 of 8survivors) and 2 of 10 survivors when DC vaccination alone was used(p<0.01 and p<0.001 respectively). Challenging similarly immunisedanimals with B. pseudomallei strain 4845 revealed a similar pattern ofresistance, with CpG matured DC immunisation giving 7 of 10 survivors,significantly better (p<0.02) than DC immunisation alone.

Challenge with strain 576 again gave a higher rate of survival inanimals immunised with CpG treated DC, with significantly improvedprotection compared with naïve controls (p<0.0001).

The ability of the DC immunisation strategy to evoke protective immuneresponses to heterologous strains of B. pseudomallei is very encouragingas the wide variety of strains found in human infections obviously havedifferent characteristics enabling induction of many different forms ofdisease and lengthy latency periods. A vaccination strategy is requiredthat will be able to protect against this wide variety of strains andinduce long lasting immunity, and a strategy based upon the use of APCwould fulfill these requirements.

However, from a practical viewpoint, this is unlikely to be applicableas a widespread vaccination strategy since each individual would requirea personalised syngeneic vaccine.

A vaccine that can stimulate DC in situ to evoke the protective immuneresponses can be produced for example by means of a formulation carryingimmunogenic reagents as described above, and in particular recombinantB. pseudomallei antigens, as well as Toll-like receptors (TLR) ligands.Binding of the TLR ligands would activate the DC initiating the processof antigen uptake, processing and presentation required in thegeneration of a protective immune response.

Killed strains of Burkholderia pseudomallei may be novel and these forma further aspect of the invention. Particular killed strains are thosedescribed above, for example wherein the strain is one which includes acapsular polysaccharide, and/or is a heat-killed strain.

In a further aspect, the invention comprises a method of preventing ortreating infection by Burkholderia pseudomallei and/or Burkholderiamallei, which method comprises administering to an animal, in particulara human, an immunogenic agent or a composition as described above.

The immunogenic agent or composition is suitably administered in thecontext of a method for preventing infection by Burkholderiapseudomallei and/or Burkholderia mallei, i.e. as a prophylactic vaccine.

In yet a further aspect, the invention provides an immunogenic agent asdescribed above for use in the preparation of a medicament for theprevention or treatment of infection by B. pseudomallei and/or B.mallei.

Preferred immunogenic agents are also as set out above.

The invention will now be particularly described by way of example withreference to the accompanying diagrammatic drawings in which:

FIG. 1 is a graph showing the survival of immunized mice followingintra-peritoneal challenge with a homologous strain of B. pseudomallei:Control mice challenged with 100MLD B. pseudomallei strain K96243(▪) orstrain 576(●); mice immunized with killed K96243 cells and challengedwith 100MLD K96243(□); and mice immunized with killed 576 cells andchallenged with 100MLD 576(∘);

FIG. 2 is a graph showing the survival of immunized mice followingintra-peritoneal challenge with a heterologous strain of B.pseudomallei: Control mice challenged with 100MLD B. pseudomallei strainK96243(▪) or strain 576(●); mice immunized with killed K96243 cells andchallenged with 100MLD K576(□); and mice immunized with killed 576 cellsand challenged with 100MLD K96243576(∘);

FIG. 3 is a graph showing the survival of mice immunized with 1E10 cellsafter intra-peritoneal challenge with B. pseudomallei strain K96243 orstrain 576: Control mice challenged with 100MLD B. pseudomallei strainK96243(▪) or strain 576(●); mice immunized with killed 1E10 cells andchallenged with 100MLD K96243(□); and mice immunized with killed 1E10cells and challenged with 100MLD 576(∘);

FIG. 4 is a graph showing the survival of control mice ●) or miceimmunized with proteinase K treated 1E10 cells (□) or proteinase Ktreated 576(∘) after intra-peritoneal challenge with 100 MLD B.pseudomallei strain 576.

FIG. 5 is a graph showing the proliferation of splenocytes from naivemice stimulated in vitro with DC pulsed with either B. pseudomalleiK96243, NCTC 4845 or strain 576. Proliferation of cells stimulated byantigen pulsed DC was greater than the proliferation of unstimulatedsplenocytes or DC alone, irrespective of which strain of heat killed B.pseudomallei was used in the assays. Each bar represents the mean of 5individuals±SD;

FIG. 6 is a graph showing proliferation of spleen cells from miceimmunised with B. pseudomallei K96243 pulsed DC, matured in the presenceor absence of CpG 1826, or administered with CpG 1826 as an adjuvant, inresponse to various strains of B. pseudomallei. The data showsignificantly enhanced proliferation in vitro of in vivo primed cells todifferent strains of B. pseudomallei, compared with unstimulated in vivoprimed cells in vitro. Each bar represents the mean of 5 individuals±SD.Statistically significant differences between immunised and controlsamples are indicated: * p<0.001, ** p<0.0001;

FIG. 7 is a graph showing numbers of cytokine secreting cells per 10⁴total spleen cells. Animals were immunised with DC matured with antigenor, antigen and CpG 1826 (6 μg ml⁻¹) or, control ODN (6 μg ml⁻¹) or DCmatured with antigen only and injected with CpG 1826 (75 μg per mouse).Maturation of DC with CpG and use of CpG as an adjuvant significantlyincreased (p<0.05) the numbers of IFN-γ secreting spleen cells comparedto the other treatment groups. No effect was seen on the numbers of IL-4secreting cells in any treatment group;

FIG. 8 is a graph showing serum antibody responses in animals immunisedwith either DC pulsed with heat killed B. pseudomallei K96243 or, DCpulsed with heat killed bacteria in the presence of CpG 1826 (6 μgml⁻¹), or DC pulsed with heat killed bacteria with CpG 1826co-administered as an adjuvant (75 μg). Each bar represents the mean of10 individuals with SEM. The CpG treated groups produced significantlymore antibody (p<0.01) than the DC only treated group.

EXAMPLE 1 Preparation and use of Heat Killed Strains of B. pseudomallei

Chemicals, Enzymes and Bacterial Strains

B. pseudomallei stains 576 and K96243 were used for this study unlessotherwise stated. B. pseudomallei strain 576 was isolated initially froma clinical case of fatal melioidosis in Thailand, B. pseudomallei strainK96243 was isolated from a 34 year old female diabetic patient in KhonKaen hospital in Thailand. All B. pseudomallei strains were cultured at37° C. in Luria Bertani (LB) broth. The capsular mutant strain of B.pseudomallei 576, termed B. pseudomallei 1E10 has been describedpreviously [T. Atkins et al. Journal Medical Microbiology, 2002, 51,539-547].

Heat Inactivation of Bacteria

B. pseudomallei strains were grown in LB at 37° C. overnight withagitation. The cultures were adjusted to the same absorbance value at590 nm with PBS and harvested by centrifugation at 5,000 rpm for 15 minsBacteria were washed once in the original culture volume of PBS,harvested again, and then resuspended in the original volume of PBS. Aviable count was performed to determine the number of bacteriaheat-killed in cfu/ml. Bacteria were heat inactivated by incubating in awater bath maintained at 80° C. for 3 hours. Inactivated bacteria werethen stored at 4° C. Inactivation was confirmed by culturing 10% of eachinactivated culture in LB for 7 days at 37° C., then plating out thebroth on LB agar plates for 7 days at 37° C. to confirm that no viablebacteria remained.

Animal Studies

Balb/c mice were age-matched, approximately six weeks old females. Stockanimals were grouped together in cages of five with free access to foodand water and subjected to a 12 h light/dark cycle. After challenge withviable B. pseudomallei, the animals were handled under bio-safety levelIII containment conditions within a half-suit isolator, compliant withBritish standard BS5726. All investigations involving animals werecarried out according to the requirements of the Animal (ScientificProcedures) Act 1986. The median lethal dose (MLD) was calculated by themethod of Reed and Muench [Am J. Hygiene, 1938, 27(3) 493-497].

An initial aim was to investigate whether immunisation with heat-killedcells could provide protection against experimental melioidosis.

Immunisation of mice with heat killed bacteria was carried out over aperiod of five weeks. Each mouse was immunised intra-peritonally (i.p.)with three injections of 100 μl killed bacteria (either strain K96243 orstrain 576) of at 1×10⁸ cfu/ml separated by two-week intervals. A periodof five weeks elapsed prior to i.p. challenge with the correspondingwild-type bacteria.

The survival of groups of 5 mice immunised with heat-killed B.pseudomallei strain K96243 and challenged 2 weeks later with 100 MLD ofstrain K96243 is summarized in the graph of FIG. 1. The control micedied within four days of challenge. However, complete protection wasseen in immunised mice up to day 12, and 80% survival was recorded 3weeks after challenge.

In the similar experiment, carried out in mice immunised with heatkilled, B. pseudomallei strain 576 and then challenged with strain 576similar results were obtained. All of the control animals died by day 4and all of the immunised animals were alive at the termination of theexperiment 3 weeks after challenge (FIG. 1.) This indicates thatheat-killed whole cells derived from different strains of B.pseudomallei can offer high levels of protection against a homologouschallenge.

EXAMPLE 2 Killed Whole Cells Protect Against B. pseudomallei StrainsExpressing Different Types of LPS

It has recently been reported that immunisation with LPS is able toprovide some protection against experimental melioidosis [M. Nelson etal supra.]. However, it seems unlikely that a single LPS type wouldprovide protection against all strains because two serologicallydistinct forms of LPS have been identified in different strains of B.pseudomallei [N. Anuntagool et al. Clinical and Diagnostic LaboratoryImmunology, 1998, 5(2) 225-229]. These different serotypes arerepresented in this study by strain K96243 (typical LPS or OPSI) andstrain 576 (atypical LPS or OPSII).

To investigate the potential for a killed whole cell vaccine to protectagainst a range of strains of B. pseudomallei we immunised mice withkilled whole cells of strain K96243 or killed whole cells of strain 576and then challenged with 100 MLD of the heterologous strain, using thesame immunization regime described in Example 1.

In both experiments (FIG. 2) the unvaccinated control mice all diedwithin 4 days. None of the immunised mice had died by the end of theexperiment (21 days post challenge). Therefore, although immunisationwith LPS can provide protection against experimental melioidosis, thereare additional protective antigens on the surface of killed cells thatprovide cross protection against strains belonging to differentserotypes.

EXAMPLE 3 The Role of Capsule in Protection

A capsular mutant of strain 576 (strain 1E10 [Atkins et al. supra]) wasused to investigate the role of capsular polysaccharide in protection.Mice were immunised with heat-inactivated B. pseudomallei strain 1E10using the immunization regime described in Example 1, and subsequentlychallenged with 100 MLD of viable B. pseudomallei strain 576 or strainK96243. The results are shown in FIG. 3.

Three weeks post challenge 60% of the immunised mice were alive whereasall of the control mice had died.

The level of protection afforded after immunisation with strain 1E10 waslower than that seen after immunisation with the encapsulated parentstrain (strain 576). However, it does appear to confirm the role ofcapsule as a protective antigen, in combination with other cellularcomponents such as protective protein antigens.

EXAMPLE 4 Investigation into the Role of Proteins in Protection

Proteinase K Digestion of Bacteria

Heat-killed suspensions (8×10⁸ cfu/ml of the bacteria described inExample 1) were centrifuged for 10 min at 13,000×g, the supernatant wasremoved and the wet weight of the pellet determined. The pellet wasre-suspended in extraction buffer (62.5 mM Tris-HCl pH 6.8, 10% v/vglycerol, 5% v/v β-mercaptoethanol and 3% w/v SDS) at a concentration of160 mg bacteria/ml buffer. The suspension was incubated at 100° C. for15 min, mixed and examined for clarity. Clear suspensions were mixedwith equal volumes of 2 mg/ml Proteinase-K solution in extraction bufferand heated at 60° C. for 2 hours. The samples were further boiled for 10min to deactivate the proteinase-K. The solution was then dialysedexhaustively against dH₂0 using Slide-A-Lyzer dialysis units (Pierce,Rockford, Ill.).

The heat-killed cells of B. pseudomallei strain 576 or strain 1E10treated with proteinase K, was then used to immunise mice using animmunization regime as described in Example 1. The mice weresubsequently challenged with 100 MLD B. pseudomallei 576 also asdescribed in Example 1. The survival rate is represented in FIG. 4.

In the control group, one mouse was alive at day 21 post challenge.Similarly, in the group immunised with proteinase K-digested 1E10, onesurvivor was remaining after 21 days. When proteinase K digested B.pseudomallei 576 cells were used for immunisation, 80% survival wasobserved 21 days post-infection. The difference between the level ofprotection after immunisation with proteinase K-treated 1E10 cells andproteinase K-treated 576 cells indicates that proteins play a role inproviding protection.

The role of each surface component can be assessed by comparing theprotection afforded by wild type and the acapsular mutant strain,summarised in Table 1.

TABLE 1 Summary of the roles of polysaccharide or protein surfaceantigens on protection against challenge with B. pseudomallei 100 MLDChallenge, Survivors at Day 21(%) Antigens K96243(typical) 576(atypical)K96243 80 100 (Typical LPS, Capsule, protein 576 100 100 (Atypical LPS,Capsule, Protein 1E10 60 60 Atypical LPS, Protein Proteinase K treatedND 80 576 Atypical LPS, Capsule Proteinase K treated ND 20 1E10 AtypicalLPS, PBS 0 0 No antigen ND means “not done”

The levels of protection seen with killed whole cells are superior tothose reported by Nelson et al. [supra] who showed that immunisationwith LPS or capsule provided 60% or 20% respectively survival at 21 dayspost infection.

The results indicate synergy between the protective responses induced byLPS and/or capsular polysaccharide and/or proteins. Preferably all threeare present for best protection, but combinations of any two, forinstance, LPS and capsule, or LPS and protein provide enhancedprotection over say LPS alone.

The findings also have important implications for the understanding ofsusceptibility to infection in human populations in areas of the worldwhere melioidosis is endemic. It is known that apparently uninfectedindividuals in these areas develop antibody responses which cross-reactwith B. pseudomallei. The responses noted here appears to provideprotection against disease.

EXAMPLE 5 Use of Immunogenic Reagents in Conjunction with DendriticCells

Methods

Experimental Animals

BALB/c mice were obtained from Charles River Ltd and maintained underSPF conditions with free access to food and water. All procedures werecarried out in accordance with the requirements of the Animals(Scientific Procedures) Act 1986.

Growth of B. pseudomallei and heat inactivation of bacteria B.pseudomallei K96243 [Holden, M. T. G., et al. Proceedings of theNational Academy of Sciences of the United States of America 2004,101(39), 14240-14245] was grown in Luria broth for 18 h at 37° C. in ashaking incubator. A viable count was obtained by culturing aliquots ofthe broth culture at 37° C. overnight on L-agar plates.

For heat killing of bacteria, broth cultures were harvested bycentrifugation and washed three times in PBS, before re-suspending inone-tenth the original volume of PBS. The bacterial cell suspension wasthen incubated in a water bath at 70° C. for 3 hours, with occasionalshaking. After inactivation the suspension was checked for viability byinoculating 10 mL volumes of L-broth with 0.5 mL aliquots of thesuspension and incubating at 37° C. for seven days. L-agar plates werethen inoculated with the total volume of the broth cultures to check forbacterial growth and incubated for a further seven days. If no growthoccurred on the agar plates the bacterial suspension was consideredinactivated.

Isolation and culture of dendritic cells from murine bone marrowProcedures were modified and developed from published techniques tooptimise the yield and viability of DC's from murine bone marrow.Briefly, bone marrow was extracted from murine rear tibiae and fibulaeand cultured at a concentration of 2×10⁶ cells mL⁻¹ in media comprisedof RPMI-1640 (Sigma, UK) supplemented with 10% heat inactivated foetalbovine serum (Sigma, UK), 1% penicillin/streptomycin/glutamine (Sigma,UK) and 50 μm 2-mercaptoethanol. The culture medium was supplementedwith 20 ng mL⁻¹ granulocyte-macrophage colony-stimulating factor, GM-CSF(R&D Systems, Europe) and 10 ng mL⁻¹ tumour necrosis factor-alpha,TNF-α(R&D Systems, Europe). Cells were cultured for 96 hours at 37° C.in the presence of 5% CO₂ in a fully humidified atmosphere, after whichtime they were removed from the culture plates by gentle scraping. Afterwashing the cell suspension was layered onto 13.7% metrizamide (w/v;Sigma, UK) and the DC purified using centrifugation.

Immunisation

DC were cultured in the presence of GM-CSF and TNF-α as described. Onceisolated and purified from culture, DC were re-suspended to 2×10⁶ cellsmL⁻¹ and pulsed with heat killed B. pseudomallei K96243 at 10⁴ cfu mL⁻¹for 18-hours at 37° C. in a humidified 5% CO₂ environment. CpG 1826(Coley Pharmaceuticals, Wellesley Mass., USA) was added to some DCcultures at a concentration of 6 μg when the cells were pulsed withantigen. The cells were then washed three times to remove anyextracellular antigen and re-suspended in sterile PBS to a concentrationof 1×10⁶ cells per 100 μL for immunisation by the intradermal (i.d.)route on day 0 and day 21 of the immunisation schedule. When used as anadjuvant concurrently with DC immunisation CpG 1826 was delivered at adose of 75 μg per mouse in sterile PBS by the intra-venous route.

Primary Response of Naïve Splenocytes

DC were cultured as described, and after washing adjusted to aconcentration of 2×10⁶ cells mL⁻¹ in culture media containing 20 ng mL⁻¹GM-CSF and 10 ng mL⁻¹ TNF-α. The cells were then incubated with heatkilled B. pseudomallei (10⁴ cfu mL⁻¹) at 37° C. in a fully humidifiedenvironment in 5% CO₂ for 18 hours.

Spleens were isolated from naïve mice humanely killed by cervicaldislocation and passed through a 70 μm nylon sieve. Red blood cells wereremoved using lysing buffer (Sigma), the remaining splenocytes werewashed, counted and re-suspended to a concentration of 5×10⁶ cells mL⁻¹.

100 μL aliquots of the splenocyte suspension were then added to 96 welltissue culture plates. Replicates of five wells were used for each ofthe test groups and controls, with 1 μg mL⁻¹ concanavalin A (Sigma, UK)being used as a positive control and culture medium as a negativecontrol.

B. pseudomallei pulsed DC were washed three times to remove anyextracellular antigen, re-suspended to the required concentration inculture medium and added to the splenocyte containing wells of the assayplates in 100 μL aliquots. The plates were then incubated at 37° C. in5% CO₂ for 96 hours.

Following the incubation period, 37 MBq of ³H-thymidine were added toeach test well on the proliferation plates and the plates re-incubatedfor a further 6 hours. Cells were then harvested onto 96-well filterplates (PerkinElmer Life Sciences) using an automated cell harvester andthe plates allowed to dry at room temperature overnight. Once dry, 20 μlof scintillation fluid (PerkinElmer Life Sciences) was added to eachwell and the plate sealed before being read on a scintillation counter(PerkinElmer Life Sciences).

Recall Response of Primed Splenocytes

Spleens from mice immunised with heat killed B. pseudomallei K96243pulsed DC were used to assess T cell recall responses to soluble B.pseudomallei K96243, NCTC 4845 or strain 576. Spleen cells, (5×10⁶ mL⁻¹in 100 μl aliquots) from immunised mice were incubated with heat killedB. pseudomallei which was added to the splenocyte cultures in 100 μLaliquots at a concentration of 10⁴ cfu mL⁻¹. The cultures were incubatedfor 72-hours in 5% CO₂ at 37° C. before addition of ³H-thymidine asdescribed above.

Enzyme Linked Immunosorbant Assay (ELISA) for Serum Antibody

Serum antibody titres to B. pseudomallei were assayed by ELISA aspreviously described [Jones, S. M., et al. Journal of MedicalMicrobiology 2002, 51(12), 1055-1062.], using heat killed B.pseudomallei K96243 as the capture antigen. Concentrations of antigenspecific IgG were determined using Ascent software (Thermo Labsystems)and the data presented as geometric mean titres with SD.

Elispot Assays for Cytokine Production

Secretion of IL-4 and IFN-γ by spleen cells from naïve and immunisedmice was examined by Elispot assay (BD Biosciences ELISPOT kits). On day35 following the primary immunisation, organs were removed from animalsculled by cervical dislocation and forced through disposable 70 μm cellstrainers (BD Biosciences) to obtain single cell suspensions. Followingcentrifugation to pellet the cells, red blood cells were removed usinglysing buffer (Sigma). The remaining cells were washed, counted andseeded onto Elispot plates (4×10⁶ cells/mL double diluted to 5×10⁵cells/mL) in medium containing heat killed B. pseudomallei K96243 at afinal concentration of 1×10⁴ cfu mL⁻¹. Four replicates were plated foreach of 5 samples per treatment group. Concanavilin A (Sigma) at a finalconcentration of 4 μg mL⁻¹ was used as a positive control. Elispotplates were incubated overnight at 37° C., 5% CO₂ in a humidifiedincubator. Assay development was performed according to the kitmanufacturer's instructions. The data are presented as means values withSD.

Challenge with B. pseudomallei

The growth of, and challenge with B. pseudomallei was performed underACDP containment level III conditions.

B. pseudomallei NCTC 4845, 576 and K96243 were grown in overnightculture as described previously and diluted to give an estimatedchallenge dose of 10⁴ cfu per mouse. Actual challenge doses weredetermined by overnight culture of inoculum samples at 37° C. on L-agarplates. Groups of 10 BALB/c mice were challenged by the intraperitoneal(i.p.) route on day 35 following primary immunisation and closelyobserved for 42 days post challenge at which point any survivors wereculled. The challenge survivors were assessed for bacterial load byculture of spleens and blood. Organs were passed through 70 μm nylonsieves into sterile PBS and blood, obtained by cardiac puncture wasdiluted 1:10 in sterile PBS. Samples were innoculated onto L-agar platesand incubated overnight at 37° C. Plates were then examined for thepresence or absence of B. pseudomallei.

Statistical Analysis

Statistical analyses were performed using the Student's paired t-testfor all in vitro experiments. Analysis of the challenge data wasperformed using PRISM graph pad survival analysis software, and p-valueswere calculated using the log rank test for trend.

Results

Proliferation Assays

Primary proliferation assays revealed that ex-vivo antigen pulsed DCwere capable of inducing proliferation in naïve mouse spleen cells (FIG.5). Secondary proliferation assays performed with spleen cells from miceimmunised with antigen pulsed DC in combination with CpG 1826 showed asignificant increase in proliferation when the CpG was used either as aconditioning agent for the DC culture or as an adjuvant with injectedDC. The effect was greatest when B. pseudomallei K96243 was used as thechallenge antigen, however there was still a significant increase inproliferation relative to the in vivo primed, unstimulated controls whenother strains of B. pseudomallei were used (FIG. 6), indicating that thedendritic cells had extracted common epitopes from the differentstrains.

Elispot Assays

Elispot assays for IL-4 and IFN-γ were performed at day 35 followingprimary immunisation. Spleen cells from animals immunised with DC pulsedwith heat killed B. pseudomallei K96243 in the presence or absence ofCpG ODN or those immunised with DC and CpG as an adjuvant were incubatedwith heat killed B. pseudomallei and the number of cytokine producingcells determined. Spleen cells from animals immunised with DC matured inthe presence of CpG 1826 produced significantly (p<0.05) more IFN-γpositive cells than the DC matured with antigen alone or those maturedwith antigen and control (non-CpG) ODN (FIG. 7).

ELISA for Serum Antibody

Analysis of serum from immunised mice for B. pseudomallei specificimmunoglobulin revealed low titres of antibody (FIG. 8) to B.pseudomallei K96243. The animals given DC matured in the presence of CpGand those given CpG as an adjuvant with an immunising dose of DC hadsignificantly higher titres than animals given DC alone (p<0.01). Thepresence of antibody reactive with B. pseudomallei strains 576 and 4845was not assayed in view of the low titres developed to the immunisingstrain.

Protection Against Challenge and Bacterial Clearance

Animals immunised with DC pulsed with B. pseudomallei K96243 either withor without CpG treatment were challenged at day 35 following primaryimmunisation, together with animals given antigen pulsed DC with CpG1826 co-injected as an adjuvant. The exact challenge doses weredetermined as; 3.8×10⁴ cfu for strain K96243, 5.1×10⁴ cfu for strain 576and 4.3×10⁴ cfu for strain 4845.

The animals immunised with DC matured in the presence of CpG 1826 showedthe highest levels of protection against all 3 challenge strains of theorganism. The use of the CpG ODN as an adjuvant with the DC immunisationalso resulted in high levels of protection (60-70%). The control CpGtreated DC and DC alone groups showed very poor levels of protection,with no better than 3 of 8 eight mice surviving in the K96243 challengedgroup (Table 2)

TABLE 2 Challenge strain/survivors Treatment group K96243 NTCC 4845Strain 576 DC + CpG in 9/10 7/10 7/10 culture DC + CpG 7/10 6/10 6/10adjuvant Control ODN 3/8  2/8  3/8  DC only 2/10 1/10 2/10 Naïve 0/8 0/8  0/8 

Challenge survival following intra-dermal immunisation with DC pulsedwith heat killed B. pseudomallei K96243, with or without CpG ODNtreatment. Challenge was with approximately 10⁴ cfu strain K96243, 4845or 576 by the intra-peritoneal route and survival at day 42 is recordedabove.

These results demonstrate that DC immunisation is capable of inducingprotective responses in immunised animals, but the addition of CpG ODNgreatly increases the levels of protection achievable.

At the end of the post challenge observation period (42 days)splenocytes and blood derived from challenge survivors were cultured at37° C. for 48 hrs on L-agar (100 μl aliquots in duplicate for eachsample). No bacterial growth was detected in any of the samples,indicating that bacterial clearance had been achieved in challengesurvivors.

The invention claimed is:
 1. An immunogenic agent which is a combinationof Burkholderia pseudomallei components, the combination of Burkholderiapseudomallei components consisting essentially of components (i), (ii)and (iii), wherein (i) is a lipopolysaccharide of Burkholderiapseudomallei, (ii) is a capsular polysaccharide of Burkholderiapseudomallei, and (iii) is an isolated immunogenic protein ofBurkholderia pseudomallei.
 2. The immunogenic agent of claim 1, whereincomponent (iii) comprises more than one isolated immunogenic protein ofBurkholderia pseudomallei.
 3. The immunogenic agent of claim 1, whereinthe isolated immunogenic protein of component (iii) is an isolatedsurface protein of B. pseudomallei.
 4. An immunogenic compositioncomprising the immunogenic agent of claim 1, in combination with apharmaceutically acceptable carrier.
 5. The immunogenic composition ofclaim 4, further comprising an adjuvant.
 6. The immunogenic compositionof claim 5, wherein the adjuvant is a moiety which activatesantigen-presenting cells.
 7. The immunogenic composition of claim 6,wherein the moiety is one which activates dendritic cells.
 8. Theimmunogenic composition of claim 7, wherein the moiety is a Toll-likereceptor ligand.
 9. The immunogenic composition of claim 6, wherein themoiety is a CpG oligonucleotide.
 10. A composition comprising theimmunogenic agent of claim 1 and antigen-presenting cells.
 11. Thecomposition of claim 10, wherein the antigen-presenting cells aredendritic cells.
 12. The composition of claim 10, further comprising anadjuvant capable of stimulating antigen-presenting cells.
 13. Thecomposition of claim 12, wherein the adjuvant is a CpG oligonucleotide.14. A method of inducing an immune response to Burkholderia pseudomalleior Burkholderia mallei in an animal, comprising administering to theanimal the immunogenic agent of claim
 1. 15. An immunogenic agentconsisting essentially of components (i), (ii) and (iii), wherein (i) isa lipopolysaccharide of Burkholderia pseudomallei, (ii) is a capsularpolysaccharide of Burkholderia pseudomallei, and (iii) is an expressionvector incorporating a nucleic acids expressing an immunogenic proteinof Burkholderia pseudomallei.
 16. The immunogenic agent of claim 15,wherein the nucleic acids encodes an isolated surface protein of B.pseudomallei.
 17. A composition consisting essentially of components(i), (ii) and (iii), wherein (i) is a lipopolysaccharide of Burkholderiapseudomallei, (ii) is a capsular polysaccharide of Burkholderiapseudomallei and (iii) is an isolated immunogenic surface protein ofBurkholderia pseudomallei or an expression vector incorporating anucleic acid expressing the immunogenic surface protein.
 18. Thecomposition of claim 17, wherein component (iii) is one or more isolatedimmunogenic surface proteins of Burkholderia pseudomallei.
 19. The ofclaim 17, wherein component (iii) is an expression vector incorporatingthe nucleic acid expressing the immunogenic surface protein.
 20. Animmunogenic composition comprising the composition of claim 17, incombination with a pharmaceutically acceptable carrier.
 21. Theimmunogenic composition of claim 20, further comprising an adjuvant. 22.The immunogenic composition of claim 21, wherein the adjuvant is amoiety which activates antigen-presenting cells.
 23. The immunogeniccomposition of claim 22, wherein the moiety is one which activatesdendritic cells.
 24. The immunogenic composition of claim 22, whereinthe moiety is a Toll-like receptor ligand.
 25. The immunogeniccomposition of claim 22, wherein the moiety is a CpG oligonucleotide.26. A composition comprising the composition of claim 17 andantigen-presenting cells.
 27. The composition of claim 26, wherein theantigen-presenting cells are dendritic cells.
 28. The composition ofclaim 26, further comprising an adjuvant capable of stimulatingantigen-presenting cells.
 29. The composition of claim 28, wherein theadjuvant is a CpG oligonucleotide.
 30. A method of inducing an immuneresponse to Burkholderia pseudomallei or Burkholderia mallei in ananimal, comprising administering to the animal the composition of claim17.
 31. An immunogenic agent which is a combination of Burkholderiapseudomallei components, the combination of Burkholderia pseudomalleicomponents consisting of components (i), (ii) and (iii), wherein (i) isa lipopolysaccharide of Burkholderia pseudomallei, (ii) is a capsularpolysaccharide of Burkholderia pseudomallei, and (iii) is an isolatedimmunogenic protein of Burkholderia pseudomallei or an expression vectorincorporating a nucleic acid expressing the immunogenic protein.
 32. Theimmunogenic agent of claim 31, wherein component (iii) is one or moreisolated immunogenic proteins of Burkholderia pseudomallei.
 33. Theimmunogenic agent of claim 31, wherein component (i) comprises an OPSIor an OPSII Burkholderia pseudomallei lipopolysaccharide.
 34. A methodof inducing an immune response to Burkholderia pseudomallei orBurkholderia mallei in an animal, comprising administering to the animalthe immunogenic agent of claim
 31. 35. The immunogenic agent of claim 1,wherein component (i) comprises an OPSI or an OPSII Burkholderiapseudomallei lipopolysaccharide.
 36. The composition of claim 17,wherein component (i) comprises an OPSI or an OPSII Burkholderiapseudomallei lipopolysaccharide.
 37. A method of inducing an immuneresponse to Burkholderia pseudomallei or Burkholderia mallei in ananimal, comprising administering to the animal the immunogenic agent ofclaim 15.